Our long-term goal is to develop therapeutic strategies that improve the survival of patients with disseminated melanoma by potentiating new and existing targeted therapies. The clinical success of BRAF- and BRAF/MEK-targeted therapy suggests that high frequency disease control may one day be a reality for genetically defined sub- groups of patients with melanoma. Despite this progress, few advances have been made in developing targeted therapeutic strategies for the 15-20% of patients whose melanomas harbor activating NRAS mutations. Although significant progress has been made in the use of immunotherapy in melanoma patients, only half of those treated respond and many of these ultimately become resistant. In NRAS-mutant melanoma the MEK inhibitor binimetinib (MEK-162) is the first targeted agent to show efficacy in patients. This drug, while showing some clinical activity, is associated with modest response rates and a median progression-free survival (PFS) of ~3 months. Research by our group and others has suggested that this limited benefit may result from the escape of limited numbers of cells that ultimately repopulate the tumor. The reasons underlying this escape are likely to be complex and are thought to result from the inherent heterogeneity of melanoma. Together these data suggest the tumor to be composed of multiple cellular populations that show differential responses to drug. The study of clonality in melanoma and the characterization of the sub-populations of cells that evade therapy represent major technical challenges and remain largely unexplored. The overarching goal of this R21 application is to define the heterogeneity of response of NRAS-mutant melanomas to MEK inhibition and to develop combination therapy strategies that target these adaptations. We will use sophisticated single cell RNA and DNA analysis techniques to characterize the clonal heterogeneity of both NRAS-mutant melanoma cell lines and patient specimens at the single cell level and will investigate how MEK inhibition alters the clonal mix. We will perform bioinformatic analyses to identify the key pathways responsible for therapeutic escape at the single cell level and will test combination therapy strategies that abrogate these in vitro and in vivo with the goal of abrogating resistance. These studies will form the groundwork for future funding applications to develop clinically relevant drug combinations.